David W. Pate

The production of cannabinoids and their associated terpenes in Cannabis is
subject to environmental influences as well as hereditary determinants. Their biosynthesis
occurs in specialized glands populating the surface of all aerial structures of the plant.
These compounds apparently serve as defensive agents in a variety of antidessication,
antimicrobial, antifeedant and UV-B pigmentation roles. In addition, the more intense
ambient UV-B of the tropics, in combination with the UV-B lability of cannabidiol, may
have influenced the evolution of an alternative biogenetic route from cannabigerol to
tetrahydrocannabinol in some varieties.

Introduction

Cannabis may have been the first cultivated plant. Records indicate use of
this crop for paper, textiles, food and medicine throughout human history (Abel 1980). It
is a dioecious annual with rather distinctive palmate leaves, usually composed of an odd
number of leaflets. Best growth occurs on recently disturbed sites of high soil nitrogen
content, so it is commonly found as a persistent weed at the edge of cultivated fields.
Mature height ranges from 1 to 5 meters, according to environmental and hereditary
dictates. Typically, the male plant is somewhat taller and more obviously flowered. These
flowers have five yellowish tepals, and five anthers that hang pendulously at maturity,
dispersing their pollen to the wind. The female plant exhibits a more robust appearance
due to its shorter branches and dense growth of leaves and flower-associated bracts. Its
double-styled flower possesses only a thin, closely adherent perianth, but is further
protected by enclosure in a cuplike bracteole (i.e., perigonal bract), subtended by a
usually monophyllous leaflet. A single achene is produced per flower and shed or dispersed
as a result of bird predation. The life cycle of the male is completed soon after
anthesis, but the female survives until full seed ripeness.

Cannabis seems a virtual factory for the production of secondary metabolic
compounds. A variety of alkanes have been identified (Adams, Jr. and Jones 1973, De Zeeuw et
al. 1973b, Mobarak et al. 1974a & 1974b), as well as nitrogenous
compounds (ElSohly and Turner 1976, Hanus 1975b), flavonoids (Gellert et al.
1974, Paris et al. 1975b, Paris and Paris 1973) and other miscellaneous compounds
(Hanus 1976a & 1976b). Terpenes appear in abundance (Hanus 1975a, Hendricks et al.
1975) and contribute to the characteristic odor of the plant (Hood et al. 1973)
and some of its crude preparations, such as hashish. The compounds which comprise the
active drug ingredients are apparently unique to this genus and are termed cannabinoids.
Cannabinoids were originally thought to exist as the phenolic compounds, but later
research (Fetterman et al. 1971a, Masoud and Doorenbos 1973, Small and Beckstead
1973, Turner et al. 1973b) has indicated their existence predominantly in the
form of carboxylic acids which decarboxylate readily with time (Masoud and Doorenbos 1973,
Turner et al. 1973b), upon heating (De Zeeuw et al. 1972a, Kimura and
Okamoto 1970) or in alkaline conditions (Grlic and Andrec 1961, Masoud and Doorenboos
1973). There are over 60 of these type compounds present in the plant (Turner et al.
1980).

Much has been published concerning the influence of heredity on cannabinoid production
(e.g., Fetterman et al. 1971b, Small and Beckstead 1973), but ecological factors
have long been thought to have an important influence by stressing the Cannabis
plant (Bouquet 1950). The resultant increased biosynthesis of the cannabinoid and terpene
containing resin, in most cases, seems likely of advantage to the organism in adapting it
to a variety of survival-threatening situations. This work reviews these biotic and
abiotic challenges and speculates on the utility of Cannabis resin to the plant.

Anatomical distribution and biogenesis of the cannabinoids

The major sites of cannabinoid production appear to be epidermal glands (Fairbairn
1972, Hammond and Mahlberg 1973, Lanyon et al. 1981, Malingre et al.
1975) which exhibit a marked variation in size, shape and population density, depending on
the anatomical locale examined. While there are no published reports of glands present on
root surfaces, most of the aerial parts possess them, along with non-glandular trichomes
(De Pasquale et al. 1974). These epidermal glands seem to fall into two broad
categories: stalked and sessile. The stalked gland (Fig. 1, front page) can consist of a
single cell or small group of cells arranged in a rosette on a single or multicellular
pedestal. Lack of thorough ontogenetic study has led to the speculation that some of this
variation may be attributable to observation of various developmental stages (Ledbetter
and Krikorian 1975). The sessile gland possesses no stalk and has secretory cells located
at or below the epidermal surface (Fairbairn 1972). In either case, the glandular cells
are covered with a "sheath" under which the resins are secreted via vesicles
(Mahlberg and Kim 1992). This sheath consists of a cuticle that coats a polysaccharide
layer (presumed cellulose) originating from the primary cell wall (Hammond and Mahlberg
1978). The resins accumulate until the sheath bulges away from the secretory cells,
forming a spheroid structure. The resin is then released by rupture of the membrane or
through pores in its surface (De Pasquale 1974). The cannabinoid content of each plant
part varies, paralleling observable gland distribution (Fetterman et al. 1971,
Honma et al. 1971a & 1971b, Kimura and Okamoto 1970, Ohlsson et al.
1971, Ono et al. 1972), although Turner et al. (1978) have disagreed.
Roots contain only trace amounts. Stalks, branches and twigs have greater quantities,
although not as much as leaf material. Vegetative leaf contains varying quantities
depending on its position on the plant: lower leaves possessing less and upper ones more.
Leaf glands are most dense on the abaxial (underside) surface. The greatest amount of
cannabinoids is found in the new growth near each apical tip (Kimura and Okamoto 1970,
Steinberg et al. 1975), although Ono et al. (1972) seem to differ on
this point. This variation in leaf gland placement may be due to either loss of glands as
the leaf matures or a greater the endowment of glands on leaves successively produced as
the plant matures. Additional study on this point is required.

Once sexual differentiation has occurred, the generation of female reproductive organs
and their associated bracts increases total plant cannabinoid content. Bracts subtending
the female flowers contain a greater density of glands than the leaves. The small cuplike
bracteole (perigonal bract) enclosing the pistil has the highest cannabinoid content of
any single plant part (Kimura and Okamoto 1970, Honma et al. 1971a & 1971b).
Second only to this is the flower itself (Fetterman et al. 1971b). Since it has
no reported epidermal gland structures, the cannabinoids present must be due to either
undiscovered production sites or simple adherence of resin from the inner surface of its
intimately associated bracteole. This conjecture is supported by the finding that the
achenes do not contain substantial amounts of the cannabinoids (Fetterman et al.
1971b, Ono et al. 1972). Reproductive structures of the male plant are also
provided with greater concentrations of the cannabinoids (Fetterman et al. 1971b,
Ohlsson et al. 1971). Stalked glands have been observed covering the tepal, with
massively stalked glands occurring on the stamen filament (Dayanadan and Kaufman 1976). In
addition, rows of very large sessile glands are found situated in grooves on the anther
itself (Dayanadan and Kaufman 1976, Fairbairn 1972) and apparently provide the pollen with
a considerable cannabinoid content (Paris et al. 1975a).

Delta-9-tetrahydrocannabinol (THC) is the cannabinoid responsible for the main
psychoactive effects of most Cannabis drug preparations (Mechoulam 1970). In some
varieties of Cannabis, additional cannabinoid homologs appear that have the usual
pentyl group attached to the aromatic ring, replaced by a propyl (De Zeeuw et al.
1972b & 1973a, Fetterman and Turner 1972, Gill 1971, Gill et al. 1970, Merkus
1971, Vree et al. 1972a, Turner et al. 1973a) or occasionally a methyl
group (Vree et al. 1971 & 1972b). Other claims have been made for butyl
(Harvey 1976) or heptyl (Isbell 1973) substitutions, but the latter announcement seems
particularly tenuous. THC is thought to be produced by the plant (Fig. 2, next page) from
cannabidiol (CBD) which, in turn, is derived from cannabigerol (CBG) generated from
non-cannabinoid precursors (Hammond and Mahlberg 1994, Shoyama et al. 1984,
Turner and Mahlberg 1988). CBG is also the biogenetic precursor of cannabichromene (CBC).
Some of the cannabinoids (e.g., cannabielsoin, cannabinol, and cannabicyclol) are probably
degradation products of the enzymatically produced cannabinoids (e.g., CBD, THC and CBC,
respectively).

Cannabinoids and environmental stress

Desiccation

THC is a viscous hydrophobic oil (Garrett and Hunt 1974) that resists crystallization
(Gaoni and Mechoulam 1971) and is of low volatility (Adams et al. 1941). Since
the sticky resins produced and exuded on the surface of the plant are varying combinations
of THC, other cannabinoids and a variety of terpenes, they can be seen as analogous to the
waxy coatings of the cacti and other succulents that serve as a barrier to water loss in
dry environments.

Bouquet (1950) has mentioned that the western side of Lebanon's mountainous Cannabis
growing areas is less favorable for resin production because of humid sea winds. De
Faubert Maunder (1976) also observed that the copious separable resin needed for hashish
production occurs only "in a belt passing from Morocco eastwards, taking in the
Mediterranean area, Arabia, the Indian sub-continent and ending in Indo-China." These
are mostly areas notable for their sparse rainfall, low humidity and sunny climate. Is it
merely coincidence that resin is produced according to this pattern, as well?

Experimental evidence is accumulating that reinforces these notions. Sharma (1975)
reported a greater glandular trichome density on leaves of Cannabis growing in
xeric circumstances. Paris et al. (1975a) have demonstrated a marked increase in
the cannabinoid content of Cannabis pollen with decreased humidity. Murari et
al. (1983) grew a range of Cannabis fiber cultivars in three climatic zones
of Italy and found higher THC levels in those plants grown in the drier
"continental" (versus "maritime") climate. Hakim et al.
(1986) report that CBD-rich English Cannabis devoid of THC produced significant
amounts of THC and less CBD, when grown in the Sudan. This trend was accentuated in their
next generation of plants.

Haney and Kutscheid (1973) have shown significant correlations of plant cannabinoid
content with factors affecting soil moisture availability: content of clay or sand,
percent slope of plot, and competition from surrounding vegetation. In some cases, this
last factor was noted to have induced a stunted plant with "disproportionally smaller
roots", which would tend to increase both the frequency and severity of desiccation
stress.

In a study of 10 Kansas locations, Latta and Eaton (1975) found wide differences in
plant cannabinoid content, observing that "delta-9-THC ranged from 0.012 to
0.49% and generally increased as locations became less favorable for plant growth,
suggesting increased plant stress enhanced delta-9-THC production." Mention
was also made of a positive correlation between competing vegetation and THC content.
Although the sampling area was not considered very moisture deficient, they speculated
that "Greater difference among locations might have been observed under drought
conditions."

Temperature

Temperature may play a role in determining cannabinoid content, but perhaps only
through its association with moisture availability. Boucher et al. (1974)
reported an increase in cannabinoid content with temperature (32o C. vs. 22o C.),
however, some variables such as increased water loss due to accelerated evaporation and
plant transpiration at high temperatures were left unaccounted. In contrast, Bazzaz et
al. (1975), using 4 Cannabis ecotypes of both tropical and temperate
character, demonstrated a definite decrease in cannabinoid production with increased
temperature (32o C. vs. 23o C.). Later studies by Braut-Boucher (1980) on clones
of 2 strains from South Africa revealed a more complex pattern of biosynthesis according
to strain, gender and chemical homologue produced. Clearly, further study of this
parameter is needed.

Soil Nutrients

Mineral balance seems to influence cannabinoid production. Krejci (1970) found
increases related to unspecified "poor soil conditions". Haney and Kutcheid
(1973) have shown the influence of soil K, P, Ca and N concentrations on Illinois Cannabis.
They report a distinctly negative correlation between soil K and plant delta-9-THC
content, although K-P interaction, N and Ca were positively correlated with it. These
minerals were also shown to affect the production of CBD, delta-8-THC and
cannabinol (CBN), although the latter two compounds are now thought to be spontaneous
degradation products of delta-9-THC. Kaneshima et al. (1973) have
demonstrated the importance of optimal Fe levels for plant synthesis of THC. Latta and
Eaton (1975) reported Mg and Fe to be important for THC production, suggesting that these
minerals may serve as enzyme co-factors. Coffman and Gentner (1975) also corroborated the
importance of soil type and mineral content, and observed a significant negative
correlation between plant height at harvest and THC levels. Interestingly, Marshman et
al. (1976) report greater amounts of THC in Jamaican plants growing in
"organically" enriched (vs. artificially fertilized) soils.

Insect predation

Wounding of the plant has been employed as a method to increase resin production
(Emboden 1972). This increase may be a response to desiccation above the point of vascular
disruption. Under natural circumstances, wounding most often occurs as a result of insect
attack. This is a source of environmental stress which the production of terpenes and
cannabinoids may be able to minimize. Cannabis is subject to few predators (Smith
and Haney 1973, Stannard et al. 1970) and has even been utilized in powdered or
extract form as an insecticide (Bouquet 1950) or repellent (Khare et al. 1974).
Its apparent defensive mechanisms include a generous covering of non-glandular trichomes,
emission of volatile terpenoid substances, and exudation of the sticky cannabinoids. Cannabis
is often noted for its aromatic quality and many of the terpenes produced are known to
possess insect-repellent properties. Among these are alpha and beta pinene, limonene,
terpineol and borneol. Pinenes and limonene comprise over 75% of the volatiles detected in
the surrounding atmosphere, but account for only 7% of the essential oil (Hood et al.
1973). Consistent with glandular trichome density and cannabinoid content, more of these
terpenes are produced by the inflorescences than the leaves, and their occurrence is also
greater in the female plant (Martin et al. 1961).

The cannabinoids may also serve as a purely mechanical defense. A tiny creature
crossing the leaf surface could rupture the tenuously attached globular resin reservoirs
of the glandular trichomes (Ledbetter and Krikorian 1975) and become ensnared in resin. A
sizable chewing insect, if able to overcome these defenses, would still have difficulty
chewing the gummy resin, along with the cystolithic trichomes and silicified covering
trichomes also present on the leaf. The utility of these epidermal features as insect
antifeedants is also inferable from their predominant occurrence on the insect-favored
abaxial leaf surface. Although the above strategies represent a seemingly sophisticated
system, many other plants (Levin 1973) and even arthropods (Eisner 1970) utilize similar
defense mechanisms, often employing identical terpenes!

Competition

Terpenes may also help to suppress the growth of surrounding vegetation (Muller and
Hauge 1967, Muller et al. 1964). Haney and Bazzaz (1970) speculated that such a
mechanism may be operative in Cannabis. They further ventured that since the
production of terpenes is not fully developed in very young plants, this may explain their
inability to compete successfully with other vegetation until more mature. The observation
(Latta and Eaton 1975) of increased THC production by plants in competition with
surrounding vegetation "at a time in the growing season when moisture was not
limiting", may indicate a stimulus for cannabinoid production beyond that of simple
water stress.

Bacteria and fungi

The cannabinoids may serve as a protectant against microorganisms. Cannabis
preparations have long served as medicines (apart from their psychoactive properties) and
are effective against a wide variety of infectious diseases (Kabelic et al. 1960,
Mikuriya 1969). These antibiotic properties have been demonstrated with both Cannabis
extracts (Ferenczy et al. 1958, Kabelic et al. 1960, Radosevic et
al. 1962) and a variety of isolated cannabinoids (ElSohly et al. 1982,
Farkas and Andrassy 1976, Gal and Vajda 1970, Van Klingeren and Ten Ham 1976). CBG has
been compared (Mechoulam and Gaoni 1965) in both "structure and antibacterial
properties to grifolin, an antibiotic from the basidiomycete Grifolia conflens."
Ferency (1956) has demonstrated the antibiotic properties of Cannabis seed, a
factor that may aid its survival when overwintering. Adherent resin on the seed surface,
as well as a surrounding mulch of spent Cannabis leaves, may serve in this
regard.

While A. alterata attacks Illinois Cannabis and destroys 2.8-45.5% of
the seed (Haney and Kutsheid 1975), the balance of these species are leaf spot diseases.
McPartland (1984) has demonstrated the inhibitory effects of THC and CBD on Phomopsis
ganjae. However, De Meijer et al. (1992), in evaluating a large collection
of Cannabis genotypes, did not find a correlation between cannabinoid content and
the occurence of Botrytis. Fungal evolution of a mechanism for overcoming the
plant's cannabinoid defenses may be responsible for their success as pathogens. Indeed,
some have been demonstrated to metabolize THC and other cannabinoids (Binder 1976, Binder
and Popp 1980, Robertson et al. 1975).

Ultraviolet radiation

Another stress to which plants are subject results from their daily exposure to
sunlight. While necessary to sustain photosynthesis, natural light contains biologically
destructive ultraviolet radiation. This selective pressure has apparently affected the
evolution of certain defenses, among them, a chemical screening functionally analogous to
the pigmentation of human skin. A preliminary investigation (Pate 1983) indicated that, in
areas of high ultraviolet radiation exposure, the UV-B (280-315 nm) absorption properties
of THC may have conferred an evolutionary advantage to Cannabis capable of
greater production of this compound from biogenetic precursor CBD. The extent to which
this production is also influenced by environmental UV-B induced stress has been
experimentally determined by Lydon et al. (1987). Their experiments demonstrate
that under conditions of high UV-B exposure, drug-type Cannabis produces
significantly greater quantities of THC. They have also demonstrated the chemical lability
of CBD upon exposure to UV-B (Lydon and Teramura 1987), in contrast to the stability of
THC and CBC. However, studies by Brenneisen (1984) have shown only a minor difference in
UV-B absorption between THC and CBD, and the absorptive properties of CBC proved
considerably greater than either. Perhaps the relationship between the cannabinoids and
UV-B is not so direct as first supposed. Two other explanations must now be considered.
Even if CBD absorbs on par with THC, in areas of high ambient UV-B, the former compound
may be more rapidly degraded. This could lower the availability of CBD present or render
it the less energetically efficient compound to produce by the plant. Alternatively, the
greater UV-B absorbency of CBC compared to THC and the relative stability of CBC compared
to CBD might nominate this compound as the protective screening substance. The presence of
large amounts of THC would then have to be explained as merely an accumulated storage
compound at the end of the enzyme-mediated cannabinoid pathway. However, further work is
required to resolve the fact that Lydon's (1985) experiments did not show a commensurate
increase in CBC production with increased UV-B exposure.

This CBC pigmentation hypothesis would imply the development of an alternative to the
accepted biochemical pathway from CBG to THC via CBD. Until 1973 (Turner and Hadley 1973),
separation of CBD and CBC by gas chromatography was difficult to accomplish, so that many
peaks identified as CBD in the preceding literature may in fact have been CBC. Indeed, it
has been noted (De Faubert Maunder 1970) and corroborated by GC/MS (Turner and Hadley
1973) that some tropical drug strains of Cannabis do not contain any CBD at all,
yet have an abundance of THC. This phenomenon has not been observed for northern temperate
varieties of Cannabis. Absence of CBD has led some authors (De Faubert Maunder
1970, Turner and Hadley 1973) to speculate that another biogenetic route to THC is
involved. Facts scattered through the literature do indeed indicate a possible
alternative. Holley et al. (1975) have shown that Mississippi-grown plants
contain a considerable content of CBC, often in excess of the CBD present. In some
examples, either CBD or CBC was absent, but in no case were plants devoid of both. Their
analysis of material grown in Mexico and Costa Rica served to accentuate this trend. Only
one example actually grown in their respective countries revealed the presence of any CBD,
although appreciable quantities of CBC were found. The reverse seemed true as well. Seed
from Mexican material devoid of CBD was planted in Mississippi and produced plants
containing CBD.

Could CBC be involved in an alternate biogenetic route to THC? Yagen and Mechoulam
(1969) have synthesized THC (albeit in low yield) directly from CBC. The method used was
similar to the acid catalyzed cyclization of CBD to THC (Gaoni and Mechoulam 1966).
Reaction by-products included cannabicyclol, delta-8-THC and delta-4,8-iso-THC,
all products which have been found in analyses of Cannabis (e.g., Novotny et
al. 1976). Finally, radioisotope tracer studies (Shoyama et al. 1975) have
uncovered the intriguing fact that radiolabeled CBG fed to a very low THC-producing strain
of Cannabis is found as CBD, but when fed to high THC-producing plants, appeared
only as CBC and THC. Labeled CBD fed to a Mexican example of these latter plants likewise
appeared as THC. Unfortunately, radiolabeled CBC was not fed to their plants, apparently
in the belief that CBC branched off the biogenetic pathway at CBD and dead ended. Their
research indicated that incorporation of labeled CBG into CBD or CBC was age dependent.
Vogelman et al. (1988) likewise report that the developmental stage of seedlings,
as well as their exposure to light, affects the occurrence of CBG, CBC or THC in Mexican Cannabis.
No CBD was reported.

Conclusions

Although the chemistry of Cannabis has come under extensive investigation,
more work is needed to probe the relationship of its resin to biotic and abiotic factors
in the environment. Glandular trichomes are production sites for the bulk of secondary
compounds present. It is probable that the cannabinoids and associated terpenes serve as
defensive agents in a variety of antidessication, antimicrobial, antifeedant and UV-B
pigmentation roles. UV-B selection pressures seem responsible for the distribution of
THC-rich Cannabis varieties in areas of high ambient radiation, and may have
influenced the evolution of an alternate biogenetic pathway from CBG to THC in some of
these strains. Though environmental stresses appear to be a direct stimulus for enhanced
chemical production by individual plants, it must be cautioned that such stresses may also
skew data by hastening development of the highly glandular flowering structures. Future
studies will require careful and representative sampling to assure meaningful results.

References

Abel E., 1980. Marihuana: The first 12,000 years. Plenum Press, New York.

Lentz P.L., C.E. Turner, L.W. Robertson and W.A. Gentner, 1974. First North American
record for Cercospora cannabina, with notes on the identification of C.
cannabina and C. cannabis. Plant Disease Reporter 58: 165-168.

Levin D.A., 1973. The role of trichomes in plant defense. Quarterly Review of Biology
48: 3-16.

Lydon J., 1985. The effects of Ultraviolet-B radiation on the growth, physiology and
cannabinoid production of Cannabis sativa L. Ph.D. Dissertation, University of
Maryland.